Article pubs.acs.org/molecularpharmaceutics
Direct Evaluation of Molecular States of Piroxicam/Poloxamer Nanosuspension by Suspended-State NMR and Raman Spectroscopies Yuki Hasegawa,† Kenjirou Higashi,† Keiji Yamamoto, and Kunikazu Moribe* Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan S Supporting Information *
ABSTRACT: A nanosuspension of piroxicam (PXC) and poloxamer 407 (poloxamer) prepared by the wet milling method was directly evaluated at the molecular level from the viewpoint of both solution and solid phases. 13C solution-state NMR measurements revealed a reduction in the concentration of dissolved poloxamer in the nanosuspension. Furthermore, the fraction of dissolved poly(ethylene oxide) (PEO) chain, which is the hydrophilic part of poloxamer, was higher than that of dissolved poly(propylene oxide) (PPO) chain, the hydrophobic part. 13C suspended-state NMR and Raman spectroscopies detected both solid-state PXC and poloxamer involved in the nanoparticles. Interestingly, the coexistence of crystalline and amorphous PXC in the nanoparticle was demonstrated. The yellow color of the nanosuspension strongly supported the existence of amorphous PXC. Changes in the peak intensity depending on the contact time in the suspended-state NMR spectrum revealed that the PEO chain of poloxamer in the nanoparticle had higher mobility compared with the PPO chain. The PEO chain should project into the water phase and form the outer layer of the nanoparticles, whereas the PPO chain should face the inner side of the nanoparticles. Amorphous PXC could be stabilized by intermolecular interaction with the PPO chain near the surface of the nanoparticles, whereas crystalline PXC could form the inner core. KEYWORDS: suspended-state NMR, nanosuspension, wet milling, nanoparticle stabilization, Raman spectroscopy
■
INTRODUCTION Nanosuspension has been developed as a promising formulation for the enhanced bioavailability of poorly watersoluble drugs.1,2 The increased surface area of these nanoparticles provides improvements in the drug dissolution rate and solubility.3,4 However, these advantages of nanosuspension could be reduced by the aggregation and agglomeration of nanoparticles.5 With the goal of stabilizing nanoparticles, ionic/ nonionic surfactants, and/or polymers are added as the stabilizer.6−8 A detailed understanding of the behavior of drug and stabilizer in nanosuspension is required for the efficient design of nanosuspension formulations. Many articles have analyzed the nanosuspension from the aspect of particle size9 by light scattering or of morphology10,11 by electron microscopy. Recently, drying-free techniques for morphology observation of suspended-nanoparticles, such as atomic force microscopy (AFM)12,13 and cryo-transmission electron microscopy (cryo-TEM),14 have been developed. However, characterizing a nanosuspension at the molecular level still poses a considerable challenge. This analytical difficulty could be derived from the composition of the nanosuspension, where dissolved components and solid nanoparticles coexist in solvent. Most spectroscopic techniques cannot always distinguish solvent, dissolved component, or solid component in the suspension, and the selective extraction © 2015 American Chemical Society
of the molecular level information on each component is hardly possible.15 NMR methods have been widely used to investigate the molecular state of both dissolved and solid components. The dissolved components in solvent are investigated by typical solution-state NMR spectroscopy. Conversely, the solid components are characterized by solid-state NMR spectroscopy according to the magic-angle spinning (MAS) technique.16 Recently, the techniques of solid-state NMR have been applied to suspended samples using the suspended-state NMR method.15,17 In this method, a sample suspension is filled into an inserted capsule and placed under MAS conditions. The cross-polarization (CP) technique, a commonly used pulse sequence of solid-state NMR spectroscopy, enables the selective observation of solid components. The suppressed molecular mobility of solid components induces the efficient CP,15,17,18 whereas the CP efficiency of solvent and dissolved components with high molecular mobility is too small to be detected. Received: Revised: Accepted: Published: 1564
December 30, 2014 March 31, 2015 April 7, 2015 April 7, 2015 DOI: 10.1021/mp500872g Mol. Pharmaceutics 2015, 12, 1564−1572
Article
Molecular Pharmaceutics Recently, suspended-state NMR methods have been applied to obtain detailed information on suspensions in various fields. Crystallinity of lactose dispersed in ethanol was revealed by suspended-state NMR measurement.19−21 This technique has also been utilized for structural analyses of proteins without sample crystallization or precipitation.22,23 In these reports, proteins sedimented to the sample tube wall under MAS conditions are detected using the CP technique. Meyer et al. proposed the use of suspended-state NMR techniques in the pharmaceutical field. In their pioneering reports, suspendedstate NMR measurement has provided detailed information on solid lipid nanoparticles15,17,24 and nanocapsules.18 We have also investigated the molecular state of a drug in a suspension of ternary nanoparticles composed of probucol/polyvinylpyrrolidone/sodium dodecyl sulfate using this technique.25 Crystalline probucol in the suspended-nanoparticles is detected in the 13 C CP/MAS NMR spectrum of the nanosuspension. Raman spectroscopy is one of the most widely used techniques for the identification and quantification of samples in gas, liquid, and solid state. Since Raman scattering is caused by the interaction between light waves and the polarizability ellipsoid of vibrating molecules, symmetric stretches and vibrations show strong bands in the Raman spectrum. Moreover, obstruction from OH vibration of solvent water is much smaller compared with infrared spectroscopy. Therefore, Raman spectroscopy is possibly a great tool for characterization of suspensions. Identification26 or quantification27 of microsized suspended drug particles has been already reported, although it is difficult to find a study in which Raman spectroscopy is applied to drug nanosuspension. In this report, preparation and detailed characterization of nanosuspensions were performed to elucidate the molecular states of a drug and stabilizer in nanosuspension. Piroxicam (PXC), a nonsteroidal anti-inflammatory drug with poor solubility, was used as a model drug. The wet milling method28 was employed to prepare the nanosuspension. Poloxamer 407 (poloxamer), a triblock copolymer composed of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), was added as a stabilizer. The micellization29 and gelation properties30 of poloxamer have attracted great attention in the pharmaceutical and material fields. Here, the direct observation of the prepared PXC/poloxamer nanosuspension was performed using AFM, NMR, and Raman techniques in addition to conventional dynamic light scattering and TEM methods. We investigated nanosuspension from the point of solution and solid phases. Dissolved components in solution phase were evaluated using the solution-state NMR method, whereas solid components of nanoparticles in the solid phase were evaluated using the suspended-state NMR and Raman spectroscopic methods. Solid-state NMR and powder X-ray diffraction measurements of freeze-dried nanosuspensions were also conducted to support the findings obtained from suspended-state NMR and Raman methods.
Figure 1. Chemical structures of (a) piroxicam (PXC) and (b) poloxamer. Carbon numbering of PXC is represented for peak assignment in 13C NMR spectra.
1:1 in a vial for 3 min. The PM was dispersed in heavy water at 100 mg/mL of PXC, followed by vortex mixing for 3 min to obtain the PM suspension. The GM suspension was prepared by wet milling of the PM suspension in a planetary ball mill PM100 (Retsch Co. Ltd., Germany) under the following conditions: ball, ZnO2; rotational speed, 400 rpm; time, 24 h. Preparation of Freeze-Dried Sample (FD). Freeze-drying of the GM suspension was carried out in a dry chamber DRC1100/freeze-dryer FDU-2100, (EYEA, Japan). Freezing was conducted at −40 °C (shelf inlet temperature) for 2 h. The ramp rate of the shelf inlet temperature was 2 min/°C. After pressure reduction for 2 h, primary and secondary drying was performed under vacuum at −20 °C for 1 h and at 20 °C for 5 h, respectively. The FDs of the PM and GM suspensions were abbreviated as FDPMSS and FDGMSS, respectively. Preparation of Amorphous PXC. Amorphous PXC was prepared by the melt/quench-cooled method. PXC was heated to 215 °C in the gas chromatograph oven GC-12A (Shimadzu, Japan). After 3 min of exposure at 215 °C, melted PXC was quench-cooled using liquid nitrogen. Amorphization of PXC was confirmed by powder X-ray diffraction measurement. Particle Size Distribution. The PM and GM suspensions were diluted 200 times followed by 3 min of sonication. The particle size distribution of each suspension was analyzed by the dynamic light scattering method using Microtrac UPA (Nikkiso Co., Ltd., Japan; measurement range, 0.0008−6.5 μm) under the following conditions: reflected light intensity, 281.4 mV; measurement time, 180 s; repeat count, 3 times; temperature, 25 °C. Atomic Force Microscopy (AFM). An atomic force microscope MPF-3D (Oxford Instruments plc, U.K.) was used to observe the morphologies of the prepared nanoparticles. First, 50 μL of 0.01% poly-L-lysine solution was dropped on mica-applied substrate, followed by drying under vacuum for 30 min. Diluted GM suspension (200 times) was then applied onto the modified substrate. After 1 day of storage at 25 °C and following washing, contact mode atomic force microscopy was performed in the liquid-environment using a cantilever TR400PSA (Olympus Corp., Japan) under the following conditions: image pixel, 1024 × 256; scanning speed, 0.5 Hz. Field Emission-Transmission Electron Microscopy (FETEM). The morphologies of nanoparticles in GM suspension were investigated using JEOL FE-TEM equipment JEM-2100F
■
MATERIALS AND METHODS Materials. PXC was purchased from Wako Pure Chemical Industries (Japan). Poloxamer 407 was supplied by BASF Japan. The chemical structures and carbon atom numbering of these materials are shown in Figure 1. All other chemicals used were of reagent grade. Preparation of Physical Mixture (PM) Suspension and Ground Mixture (GM) Suspension. The PM of PXC with poloxamer was prepared by vortex mixing at a weight ratio of 1565
DOI: 10.1021/mp500872g Mol. Pharmaceutics 2015, 12, 1564−1572
Article
Molecular Pharmaceutics (JEOL, Japan) with an applied voltage of 120 kV. Collodion film-supported grid Cu200 (Nisshin EM Co. Ltd., Japan) was exposed to hydrophilic treatment for 1 min. A diluted GM suspension (200 times) was loaded onto the film for 1 min following negative staining using 3% phosphotungstic acid solution (pH 7.4) for 1 min. FE-TEM measurement was performed after drying the film in a desiccator for 1 day. Determination of Dissolved PXC Concentration by HPLC. Sample suspensions were ultracentrifuged at 1.5 × 105g at 25 °C for 1 h. The PXC concentrations in these supernatants were determined using HPLC measurements. Sample solutions were diluted with acetonitrile before quantification and applied to SUPERIOREX ODS column (5 μm, 150 mm × 4.6 mm, Shiseido, Japan) at 40 °C. The mobile phase consisted of 1/1 acetonitrile and phosphate solution (pH 2.2). The PXC peak on HPLC chart was detected at λ = 245 nm, and the retention time was approximately 4 min. The measurements were repeated 3 times using freshly prepared samples. Solution-State NMR Spectroscopy. All solution-state NMR measurements were performed using a JEOL Resonance ECA-500 system (JEOL Resonance, Japan) with a magnetic field of 11.7 T. The PM and GM suspensions prepared with heavy water were transferred into 5 mm NMR sample tubes. Chemical shifts were referenced to an internal signal of 5 mg/ mL of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) at 0.0 ppm. 1H NMR spectra were obtained at 25 °C with 15 Hz of sample spinning. A pulse delay time of 10 s and a scan of 256 accumulations were used. The measurements were repeated 3 times using freshly prepared samples. Solid-State and Suspended-State NMR Spectroscopy. All 13C solid-state and suspended-state NMR spectra were obtained using the JEOL Resonance ECA-600 NMR spectrometer. Samples (ca. 100 mg for powder and 20 μL for suspension samples) were placed into 4 mm zirconium rotors. 13 C spectra were acquired by CP/MAS/total spinning sideband suppression (TOSS) together with a high-power 1H decoupling at 5 kHz of spinning speed under an inlet air temperature at 25 °C. For each spectrum, a total accumulation number of 30,000 was acquired. Pertinent acquisition parameters included pulse delay times of 2 and 15 s, a CP contact time of 0.5−10 ms and a 1H 90° pulse of 3.6 μs. All spectra were externally referenced to tetramethylsilane by setting the methine peak of hexamethylbenzene to 17.3 ppm.
Figure 2. Particle size distribution patterns (left) and sample appearances (right) of (a) physical mixture (PM) suspension, (b) ground mixture (GM) suspension, and (c) GM suspension after storage for 1 month at 25 °C.
suspended state. AFM topographical image of GM suspension showed nanosized particles (Figure 3a). The height graph
■
Figure 3. Atomic force microscopy (AFM) (a) topographical image and (b) height graph under the dotted line in topographical image.
RESULTS AND DISCUSSION Preparation of PXC/Poloxamer Nanosuspension. Figure 2 shows the particle size distribution patterns and sample appearance of PM and GM suspensions. In the case of PM suspension, it is impossible to measure the dynamic light scattering because of the precipitation of PXC crystal just after the preparation (Figure 2a). In contrast, GM suspension showed a nano-ordered mean particle size of approximately 232 nm (Figure 2b). The unimodal distribution of particles in GM suspension was maintained during the storage at 25 °C for 1 month (Figure 2c). These results clearly demonstrated that the wet milling of PXC and poloxamer provided a stable nanosuspension. It should be noted that the color of the suspension was changed from white (PM suspension) to yellow (GM suspension). This color change should reflect the change of molecular state of PXC, as indicated by Sheth et al.31 Morphology Observation of PXC/Poloxamer Nanoparticles by AFM and FE-TEM. AFM was used to investigate the morphologies of PXC/poloxamer nanoparticles in the
under the dotted line shows that the height of the particles was approximately 200 nm, which was in agreement with the mean particle size obtained from dynamic light scattering (Figure 3b). FE-TEM measurements were carried out for the further characterization of nanoparticles in GM suspension (Figure 4). The FE-TEM images showed block-shaped nanoparticles with sizes of 100−200 nm, confirming that nanosized particles were obtained. Solution Phase Evaluation of PXC/Poloxamer Nanosuspension. The concentration of dissolved PXC in the supernatant was determined by HPLC after 1 h of ultracentrifugation at 1.5 × 105g (Table 1). The PXC crystal barely dissolved in water, with a solubility of 26 μg/mL at 25 °C. Further, the PXC concentration in the PM and GM suspensions increased at 169 and 281 μg/mL, respectively. Poloxamer, an amphiphilic block copolymer, forms micelles above the critical micelle concentration (7 mg/mL) in aqueous 1566
DOI: 10.1021/mp500872g Mol. Pharmaceutics 2015, 12, 1564−1572
Article
Molecular Pharmaceutics
The calibration curve was prepared for each peak of PEO and PPO methylene group using a poloxamer solution as a standard. The fractions of dissolved PEO and PPO chains in poloxamer against the loaded concentration of PM suspension were determined as 97.8% and 98.1%, respectively (Table 2). Table 2. Fraction of Dissolved PEO and PPO Chains in Poloxamer against the Loaded Concentration (100 mg/mL) Determined by 13C NMR Measurement (n = 3, mean ± SD)
Figure 4. Field emission-transmission electron microscopy (FE-TEM) images of GM suspension magnified at (a) ×50,000 and (b) ×200,000.
Table 1. PXC Concentration of Supernatant of PM and GM Suspensions after Ultracentrifugation at 1.5 × 105g for 1 h Determined by HPLC (n = 3, mean ± SD); the Preloaded Concentration of PXC in Each Sample Was 100 mg/mL
PM suspension GM suspension
PXC conc. (μg/mL) water PM suspension GM suspension
dissolved PEO (%)
dissolved PPO (%)
97.8 ± 1.9 91.5 ± 2.2
98.1 ± 3.0 78.2 ± 2.8
Both the PEO and PPO chains of poloxamer in PM suspension were sufficiently mobile to be reflected in solution-state NMR spectra, and almost all of the poloxamer was dissolved as polymer micelle. The fractions of dissolved PEO and PPO chains in GM suspension were reduced to 91.5% and 78.2%, respectively. Lennart et al. reported that neither a felodipine nor a miglyol peak in the nanosuspension appears in the 1H NMR spectrum because felodipine and miglyol form solidamorphous nanoparticles.39 The reduced concentration of poloxamer in GM suspension also showed that some poloxamer molecules participated in the nanoparticles. Furthermore, the different fractions of PEO and PPO in the GM suspension demonstrated the different surrounding environment of each methylene chain within a poloxamer molecule. In the case of nanosuspension composed of polylactic acid (PLA)−polyethylene glycol (PEG) copolymer with core− shell structure, the peaks of the PEG outer layer with high mobility were clearly observed in the solution-state 1H NMR spectrum, whereas those of PLA core with solid-like mobility are not found.40 Hence, the greater concentration of PEO compared with that of PPO observed in the GM suspension could reflect the formation of core−shell nanoparticles. Hydrophilic PEO chain should form the outer layer of the particles facing the water phase, whereas hydrophobic PPO chain should exist near the inner core of the particles. Solid Phase Evaluation of PXC/Poloxamer Nanosuspension. Prior to the suspended-state NMR measurement, the physical stability of PXC/poloxamer nanosuspension under MAS conditions was examined. The time-consuming measurement under strong centrifugation force requires the superior stability of the nanoparticles.17,22 To evaluate the influence of centrifugal force on the size of the nanoparticles, the GM suspension was spun at 5 kHz in an NMR sample tube (ϕ = 4 mm) for 3 days. Although the sedimentation of nanoparticles onto the tube wall was observed after the duration, redispersion was achieved by 3 min of sonication. The redispersed suspension showed the mean particle size of 226 nm with the unimodal distribution (Figure S1). The NMR measurement of sedimented sample under the MAS condition has been established as one of the effective tools for the structural analysis of protein solution.22,23 The redispersibility of sedimented proteins after MAS is the critical requirement in this analysis. Given the eminent ability of redispersion, the experimental condition on suspended-state NMR measurement with 5 kHz for 3 days was suitable for evaluating the GM suspension. The solid phase of PXC/poloxamer nanosuspension was selectively evaluated by 13C CP/MAS NMR measurements shown in Figure 6. In the spectrum of PXC crystal (Figure 6a),
26.1 ± 1.1 169.3 ± 5.2 281.4 ± 14.6
environments.32 Many articles have focused on the solubilization of hydrophobic drugs by poloxamer micelles.33−35 The apparent solubility of PXC improved considerably when poloxamer concentration increased.36 Therefore, an enhanced PXC concentration in the PM suspension should be derived from solubilization by poloxamer micelles. The even higher PXC concentration in the GM suspension might be explained by the size of nanoparticles; some nanoparticles below 100 nm remained in the supernatant, even after the ultracentrifugation. In either case, the dissolved PXC represented less than 1% of the loaded concentration (100 mg/mL) in both the PM and GM suspensions; nearly none of the PXC dissolved, instead existing as a solid in both suspensions. Further investigation of the solution phase was conducted by solution-state NMR, which detects the components with high mobility, i.e., dissolved components. Figure 5 shows the 13C
Figure 5. 13C solution-state NMR spectra of (a) PM suspension and (b) GM suspension at 25 °C.
solution-state NMR spectra of PM and GM suspensions. PXC peaks were rarely found in both spectra because of its quite low concentration, i.e., below 300 μg/mL. Conversely, PPO methyl, methylene, methine, and PEO methylene peaks in poloxamer were observed at 19.6, 72.6, 74.1, and 73.5 ppm, respectively.37,38 Quantitative analysis of dissolved PEO and PPO in poloxamer was carried out by comparing the peak area of the methylene group with that of DSS in the 13C NMR spectra. 1567
DOI: 10.1021/mp500872g Mol. Pharmaceutics 2015, 12, 1564−1572
Article
Molecular Pharmaceutics
Figure 7. Expansion of 13C CP/MAS NMR spectra between 100 and 180 ppm of (a) PXC crystal, (b) PM suspension, and (c, d) GM suspension. Panels a−c and d were obtained with pulse delay times of 15 and 2 s, respectively.
Figure 6. 13C cross-polarization/magic-angle spinning (CP/MAS) NMR spectra of (a) PXC crystal, (b) poloxamer (solid), (c) poloxamer solution, (d) PM suspension, and (e) GM suspension.
the existence of highly disordered PXC, presumably amorphous PXC, in the GM suspension.43 Generally, a crystal in which molecules are arranged regularly has a longer spin−lattice relaxation time (T1) compared with amorphous because the mobility of a solid component falls into the slow motional regime below the T1 minimum. Lubach et al. selectively detected an amorphous component from a mixture of crystalline and amorphous components by applying appropriate pulse delay times (PD) (T1amorphous < PD < T1crysal).44 We followed this method for the closer investigation of the PXC molecular state in GM suspension. The 1H-T1 value of PXC crystal was determined to be 12.7 s by 1H-T1 measurements using the inversion recovery method. The 13C CP/MAS NMR spectrum of GM suspension obtained using considerably shorter PD (2 s) compared with the 1H-T1 (12.7 s) showed further broadening of PXC peaks and a greater line half-width of Ci at 282.5 Hz (Figure 7d). These features could be interpreted to show that amorphous PXC was emphasized in this spectrum. Thus, it was concluded that the CP/MAS NMR spectrum of the GM suspension at a PD of 15 s (Figure 7c) involved both sharp and broad peaks that corresponded to crystalline and amorphous PXC in GM suspension, respectively. Amorphous PXC was prepared by the melt/quench-cooled method to confirm the existence of amorphous PXC in the GM suspension (Figure S2).45 The amorphous PXC was yellow in color, coinciding with the pale yellow of GM suspension. Unfortunately, the CP/MAS NMR measurement requires a long duration under MAS and was thus difficult to apply due to the poor stability of amorphous PXC.31 The molecular mobility of poloxamer in GM suspension was evaluated by 13C CP/MAS NMR measurements by varying the contact time (τct). The τct dependency of methylene peaks of PEO and PPO are shown in Figure 8a. The CP build-up curves, where the peak intensities in each spectrum were normalized to the maximum, were plotted as a function of τct (Figure 8b). The methylene peak intensity of PPO reached its maximum at τct 1 ms and decayed rapidly. The speedy decay of methylene peak of PPO after the rapid build-up could reflect considerably short 1 H and/or 13C T1ρ. It could be deduced that the PPO chain on the nanoparticle surface in water should be rather mobile, compared with that in powder form. Meanwhile, the intensity of PEO methylene peak showed continuous increase until 10 ms. The slower buildup of PEO peak comparing to that of PPO
peaks of methyl and aromatic carbon appeared at 38 and 100− 180 ppm, respectively.31,41 The spectrum of poloxamer in solidstate (Figure 6b) showed the peaks of PEO methylene carbon at 72.6 ppm and PPO methylene and methine carbon at 73.5 and 74.1 ppm, respectively.37,38,42 In contrast, no peak was observed in the spectrum of poloxamer solution (Figure 6c). Because the CP method employs magnetic transfer via dipolar interaction, dissolved components where the dipolar interactions average to zero are not detected.19 PXC was the sole component detected in the suspended-state NMR spectrum of the PM suspension, whereas both PXC and poloxamer appeared in the spectrum of the GM suspension (Figure 6d,e). The absence of poloxamer peak in the CP/MAS NMR spectra of the PM suspension was interpreted as evidence that almost all of the poloxamer was dissolved (Table 2). Mayer et al. reported that the poloxamer peaks are clearly observed with polybutylcyanoacrylate (PBC) peaks in the suspended-state NMR spectrum of a PBC/poloxamer nanocapsule suspension because the restricted mobility of poloxamer absorbed on nanocapsule enables the efficient CP.24 Thus, the poloxamer peak in the spectrum of the GM suspension could reflect the poloxamer molecules with restricted mobility on the surface of the nanoparticles. Quantitative analysis by solution-state 13C NMR spectroscopy, as described in Table 2, showed that the mobility of some poloxamer molecules was suppressed because of their involvement in the nanoparticles in the GM suspension. These poloxamer molecules should appear in the CP/MAS spectrum of the GM suspension as solid phase. For further investigation of the molecular state of PXC, the 13 C CP/MAS NMR spectra were expanded (Figure 7). The chemical shift and peak shape of PXC in the spectrum of the PM suspension were similar to those of the PXC crystal, indicating that PXC existed as crystal in the PM suspension. Conversely, the peaks of PXC were broadened in the spectrum of the GM suspension (Figure 7c). The line half-width of carbonyl carbon Ci at 167.4 ppm was considerably greater in the GM suspension (at 224.8 Hz) compared with those in PXC crystal (145.6 Hz) and the PM suspension (166.7 Hz). Moreover, the relative peak intensities of Ca, Cb, and Ck against other peaks were remarkably decreased in the spectrum of the GM suspension. These changes of peak shape could indicate 1568
DOI: 10.1021/mp500872g Mol. Pharmaceutics 2015, 12, 1564−1572
Article
Molecular Pharmaceutics
Figure 8. (a) Expansion of 13C CP/MAS NMR spectra between 60 and 80 ppm obtained at different contact times and (b) 1H−13C CP build-up curves of methylene carbon peaks of PEO (⧫) and PPO (□) in the spectrum of the GM suspension.
Figure 9. Raman spectra of (a) PXC crystal, (b) PXC amorphous, (c) poloxamer (solid), (d) poloxamer solution, (e) PM suspension, and (f) GM suspension.
should reflect the solid phase poloxamer involved in the nanoparticles. Moreover, the PXC peaks of δSO2 and νC−N were broadened, indicating the existence of amorphous PXC in the GM suspension. Evaluation of FD of PXC/Poloxamer Nanosuspension. GM suspension was freeze-dried to confirm the molecular state of PXC. The FDGMSS suspension showed a unimodal particle size distribution, with a mean particle size of ca. 248 nm (Figure S3). This particle size was almost comparable to that of the GM suspension (ca. 232 nm), suggesting that the properties of the GM suspension were maintained in the process of freezedrying. It is noteworthy that the FDGMSS was pale yellow in color, similar to that of the GM suspension. PXRD measurements were performed to deduce the crystallinity of PXC in FDGMSS. Characteristic diffraction peaks were observed at 2θ = 8.5, 14.4, 17.6, and 27.3° in the PXRD pattern of PXC crystal, whereas a halo pattern was observed in that of amorphous PXC (Figure 10a,b). Poloxamer, which is classified as semicrystal, showed the diffraction patterns at 2θ = 19.4 and 23.5° (Figure 10c). These peaks of crystalline PXC and poloxamer were clearly observed in the PXRD patterns of FDPMSS and FDGMSS, thus confirming the existence of crystalline PXC in PM suspension and GM suspension (Figure 10d,e). It should be noted that the
indicated that magnetic transfer could occur less efficiently in the PEO than in the PPO. The CP efficiency reflects local 1 H−13C interactions such as dipolar interactions, and carbons that have more 1H nearby are favorable for CP.46 In contrast, carbons with high mobility are unfavorable for CP because the molecular motion slows CP efficiency. Therefore, the lower CP efficiency in the PEO methylene peak compared with the equivalent PPO indicated that the PEO chain could have further higher mobility than the PPO chain. A portion of poloxamer molecules should exist on the surface of nanoparticles, thus contributing to their stable dispersion. From the τct dependency study in Figure 8, the PEO chain should be relatively mobile covering the nanoparticles, whereas PPO chain is fixed on their surface. The quantitative analysis in Table 2 demonstrating the greater fraction of PEO compared with PPO is in agreement with this mobility difference. The proposed structure could be well acceptable because the PEO chains are relatively more hydrophilic than PPO chains. Poloxamer, which is a triblock copolymer composed of PEO−PPO−PEO, should form a U-shaped structure that directs the PPO chain to the nanoparticle surface.47 The outer layer of PEO chain could prevent the aggregation and agglomeration of nanoparticles by steric repulsion. Figure 9 shows the Raman spectra between 1300 and 1700 cm−1. The Raman peaks of the asymmetry deformation vibration of the sulfonyl group (δSO2, 1346 cm−1), deformation vibration of the methyl group (δCH3, 1434 cm−1), stretching vibration of C−N (νC−N, 1524 cm−1), and carbonyl (νCO, 1596, 1606 cm−1) in amide group were observed in the spectrum of PXC crystal (Figure 9a).41,48 The peaks of δSO2 and νC−N were broadened by the amorphization of PXC (Figure 9b). The broad peak at 1482 cm−1 in the spectrum of solidstate poloxamer reflected the deformation vibration of methylene (δCH2).49,50 In contrast, no peaks were observed in the Raman spectrum of poloxamer solution, indicating that the Raman scattering from the dissolved poloxamer was too weak to be detected at this concentration (100 mg/mL) (Figure 9d). The PXC peaks in the spectrum of PM suspension appeared at the same wavenumber as those of PXC crystal, indicating that PXC existed as crystal (Figure 9e). The δCH2 peak of poloxamer was not found due to its complete dissolution. Conversely, peaks of both PXC and poloxamer appeared in the spectrum of GM suspension (Figure 9f). The δCH2 peak of poloxamer
Figure 10. Powder X-ray diffraction (PXRD) patterns of (a) PXC crystal, (b) amorphous PXC, (c) poloxamer (solid), (d) FDPMSS, and (e) FDGMSS. 1569
DOI: 10.1021/mp500872g Mol. Pharmaceutics 2015, 12, 1564−1572
Article
Molecular Pharmaceutics
Recently, it has been reported that the physical properties and bioavailability of nanosuspension are significantly affected by the drug crystal form or its crystallinity.51 Crystalline nanoparticles exhibit the same shape as unprocessed crystalline drug,52 whereas amorphous nanoparticles show spherical morphology.14 The amorphous felodipine nanoparticles are highly unstable in the presence of crystalline particles because they have a higher saturation solubility than do crystalline nanoparticles.11 The method used to prepare the amorphous nanoparticles could also affect the stability and supersaturation of the itraconazole nanosuspension.53 Furthermore, Yang et al. reported that amorphous itraconazole nanoparticles exhibited higher Cmax and AUC0−24 than did crystalline nanoparticles in systemic circulation because of the former’s rapid dissolution and sustained supersaturation.54 The evaluation of nanosuspension in those studies, however, was primarily based on their size and morphology, and the molecular state of drug was indirectly evaluated by PXRD and differential scanning calorimetry measurements after the freeze-drying process. Although Lindfors et al. demonstrated a quantitative analysis of amorphous, crystalline, and dissolved felodipine using a simple turbidimetric measurement,14 this method might not always be applicable to other drugs. However, NMR and Raman spectroscopies enable the direct evaluation of nanosuspensions, as shown in this study, thus revealing the molecular state of both the stabilizer and the drug. Because of this advantage, the real-time monitoring of crystallinity changes of suspended nanoparticles in storage is possible and gives detailed information on the stabilization mechanism of nanosuspension. Furthermore, the revealed molecular states of nanosuspensions can potentially clarify the mechanisms of drug solubility and bioavailability enhancement.
diffraction intensity of the PXRD patterns of FDGMSS was nearly half of the FDPMSS. Molecular arrangement disorder and size reduction result in the reduction of peak intensity in the PXRD pattern. The reduced peak intensity of PXC in FDGMSS should reflect the partial amorphization of PXC and nanoparticle formation with a size of ca. 250 nm. The 13C CP/MAS NMR spectra of FDs are expanded in Figure S4, showing the molecular state of PXC. The shapes and chemical shifts of PXC peaks in the spectrum of FDPMSS were comparable to those of the PXC crystal, thus confirming the existence of crystalline PXC in the PM suspension (Figure S4b). The spectrum of FDGMSS also showed crystalline PXC peaks, although they were overlapped by broadened peaks of amorphous PXC. The line half-width of carbonyl carbon Ci (167.4 ppm) was much greater in FDGMSS (200.9 Hz) compared with that of PXC crystal (145.6 Hz) and FDPMSS (154.2 Hz). Reductions of the relative peak intensities of Ca, Cb, and Ck were also observed. These spectral features clearly demonstrated the coexistence of crystalline and amorphous PXC in GM suspension. Speculated Structure of PXC/Poloxamer Nanosuspension. The proposed structure of GM suspension based on this study is represented in Figure 11. Most of the poloxamer
■
CONCLUSIONS
A PXC/poloxamer nanosuspension with a particle size of ca. 230 nm was successfully prepared by the wet milling process. The solution-state NMR study indicated the formation of core−shell nanoparticles in the prepared nanosuspension. The direct evaluation of the solid phase by a combination of suspended-state NMR and Raman spectroscopies revealed the molecular states of PXC and poloxamer, enabling us to illustrate the structure of the nanosuspension. The mobility difference between PEO and PPO that was revealed by the τct dependency study suggested that the mobile PEO chain should form the outer layer of the nanoparticles, whereas the PPO should be strongly associated with the drug near the surface. The coexistence of crystalline and amorphous PXC was confirmed by a close investigation of PXC crystallinity in suspended and freeze-dried nanoparticles. This amorphous PXC could be localized near the surfaces of nanoparticles and interact with a PPO chain of the poloxamer. This study clearly demonstrated a direct evaluation of the molecular states of drug and stabilizer in the nanosuspension by means of advanced spectroscopic techniques. Further study at the molecular level will allow us to deduce detailed structures of various nanosuspensions, which is critical information for designing new nanosuspension formulations. We also expect that this study will help further the progress of quality assessment and the development of nanosuspension formulations.
Figure 11. Schematic representation of GM suspension.
formed micelles in solution phase, whereas other poloxamers participated in the nanoparticle as solids. These poloxamer molecules covered the surface of nanoparticles by forming Ushaped structures. Here, the PPO chain with restricted mobility strongly associated with the surface of PXC nanoparticles, whereas the hydrophilic PEO chain formed the outer layer of nanoparticles with high mobility. This outer layer of the PEO chain prevented the aggregation and agglomeration of nanoparticles via steric repulsion. The spectral analysis described above strongly suggests the coexistence of crystalline and amorphous PXC in GM suspension. Considering the poor stability of amorphous PXC,31 the PPO chain fixed on the nanoparticle’s surface could contribute to maintain the amorphous state of PXC in water via an intermolecular interaction. Meanwhile, the inner core of the nanoparticle, where no poloxamer existed, should be formed by crystalline PXC. The PXC peaks in the 13C suspended-state NMR and Raman spectra of GM suspension were not obviously the sums of broad and sharp peaks and could be corresponded to a continuum of the amorphous and crystalline states. Therefore, the molecular state of PXC could change continuously from the amorphous state at the nanoparticle surface to the crystalline state at the inner core. 1570
DOI: 10.1021/mp500872g Mol. Pharmaceutics 2015, 12, 1564−1572
Article
Molecular Pharmaceutics
■
(11) Lindfors, L.; Skantze, P.; Skantze, U.; Westergren, J.; Olsson, U. Amorphous Drug Nanosuspensions. 3. Particle Dissolution and Crystal Growth. Langmuir 2007, 23 (19), 9866−9874. (12) Moribe, K.; Wanawongthai, C.; Shudo, J.; Higashi, K.; Yamamoto, K. Morphology and Surface States of Colloidal Probucol Nanoparticles Evaluated by Atomic Force Microscopy. Chem. Pharm. Bull. 2008, 56 (6), 878−880. (13) Moribe, K.; Ogino, A.; Kumamoto, T.; Ishikawa, T.; Limwikrant, W.; Higashi, K.; Yamamoto, K. Mechanism of Nanoparticle Formation from Ternary Coground Phenytoin and Its Derivatives. J. Pharm. Sci. 2012, 101 (9), 3413−3424. (14) Lindfors, L.; Forssén, S.; Skantze, P.; Skantze, U.; Zackrisson, A.; Olsson, U. Amorphous Drug Nanosuspensions. 2. Experimental Determination of Bulk Monomer Concentrations. Langmuir 2005, 22 (3), 911−916. (15) Mayer, C. Nuclear Magnetic Resonance on Dispersed Nanoparticles. Prog. Nucl. Magn. Reson. Spectrosc. 2002, 40 (4), 307−366. (16) Paudel, A.; Geppi, M.; Van Den Mooter, G. Structural and Dynamic Properties of Amorphous Solid Dispersions: The Role of Solid-State Nuclear Magnetic Resonance Spectroscopy and Relaxometry. J. Pharm. Sci. 2014, 103 (9), 2635−2662. (17) Mayer, C. NMR Studies of Nanoparticles. In Annual Reports on NMR Spectroscopy; Webb, G. A., Ed.; Academic Press: New York, 2005; Vol. 55, pp 205−258. (18) Mayer, C.; Hoffmann, D.; Wohlgemuth, M. Structural Analysis of Nanocapsules by Nuclear Magnetic Resonance. Int. J. Pharm. 2002, 242 (1−2), 37−46. (19) Crisp, J. L.; Dann, S. E.; Edgar, M.; Blatchford, C. G. The In-Situ Solid-State NMR Spectroscopy Investigation of Alcoholic Lactose Suspensions. Solid State Nucl. Magn. Reson. 2010, 37 (3−4), 75−81. (20) Bertini, I.; Engelke, F.; Gonnelli, L.; Knott, B.; Luchinat, C.; Osen, D.; Ravera, E. On the Use of Ultracentrifugal Devices for Sedimented Solute NMR. J. Biomol. NMR 2012, 54 (2), 123−127. (21) Bertini, I.; Luchinat, C.; Parigi, G.; Ravera, E. SedNMR: On the Edge between Solution and Solid-State NMR. Acc. Chem. Res. 2013, 46 (9), 2059−2069. (22) Bertini, I.; Luchinat, C.; Parigi, G.; Ravera, E.; Reif, B.; Turano, P. Solid-State NMR of Proteins Sedimented by Ultracentrifugation. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (26), 10396−10399. (23) Gardiennet, C.; Schütz, A. K.; Hunkeler, A.; Kunert, B.; Terradot, L.; Böckmann, A.; Meier, B. H. A Sedimented Sample of a 59 kDa Dodecameric Helicase Yields High-Resolution Solid-State NMR Spectra. Angew. Chem., Int. Ed. 2012, 51 (31), 7855−7858. (24) Hoffmann, D.; Mayer, C. Cross Polarization Induced by Temporary Adsorption: NMR Investigations on Nanocapsule Dispersions. J. Chem. Phys. 2000, 112 (9), 4242−4250. (25) Zhang, J.; Higashi, K.; Limwikrant, W.; Moribe, K.; Yamamoto, K. Molecular-Level Characterization of Probucol Nanocrystal in Water by In Situ Solid-State NMR Spectroscopy. Int. J. Pharm. 2012, 423 (2), 571−576. (26) Doub, W.; Adams, W.; Spencer, J.; Buhse, L.; Nelson, M.; Treado, P. Raman Chemical Imaging for Ingredient-Specific Particle Size Characterization of Aqueous Suspension Nasal Spray Formulations: A Progress Report. Pharm. Res. 2007, 24 (5), 934−945. (27) Park, S. C.; Kim, M.; Noh, J.; Chung, H.; Woo, Y.; Lee, J.; Kemper, M. S. Reliable and Fast Quantitative Analysis of Active Ingredient in Pharmaceutical Suspension Using Raman Spectroscopy. Anal. Chim. Acta 2007, 593 (1), 46−53. (28) Shegokar, R.; Müller, R. H. Nanocrystals: Industrially Feasible Multifunctional Formulation Technology for Poorly Soluble Actives. Int. J. Pharm. 2010, 399 (1−2), 129−139. (29) Alexandridis, P. Poly(ethylene oxide)/Poly(propylene oxide) Block Copolymer Surfactants. Curr. Opin. Colloid Interface Sci. 1997, 2 (5), 478−489. (30) Bentley, M. V. L. B.; Marchetti, J. M.; Ricardo, N.; Ali-Abi, Z.; Collett, J. H. Influence of Lecithin on Some Physical Chemical Properties of Poloxamer Gels: Rheological, Microscopic and In Vitro Permeation Studies. Int. J. Pharm. 1999, 193 (1), 49−55.
ASSOCIATED CONTENT
S Supporting Information *
Particle size distribution patterns and sample appearances of GM suspension before and after spinning at 5 kHz for 3 days (Figure S1); PXRD pattern and sample appearance of PXC amorphous (Figure S2); Particle size distribution patterns and sample appearances of GM and FDGMSS suspensions (Figure S3); Expansion of 13C CP/MAS NMR spectra between 100− 180 ppm of PXC crystal, FDPMSS, and FDGMSS (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Tel: +81-43-226-2865. Fax: +81-43-226-2867. E-mail:
[email protected]. Author Contributions †
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We appreciate Dr. Katsuhide Terada in Toho University and Dr. Etsuo Yonemochi in Hoshi Pharmaceutical University for their kind help on Raman spectroscopy measurement. We would like to thank BASF Japan for their gift of poloxamer. This study was supported in part by a Grant-in Aid from the Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho) of Japan.
■
REFERENCES
(1) Chen, H.; Khemtong, C.; Yang, X.; Chang, X.; Gao, J. Nanonization Strategies for Poorly Water-Soluble Drugs. Drug Discovery Today 2011, 16 (7−8), 354−360. (2) van Eerdenbrugh, B.; van den Mooter, G.; Augustijns, P. TopDown Production of Drug Nanocrystals: Nanosuspension Stabilization, Miniaturization and Transformation into Solid Products. Int. J. Pharm. 2008, 364 (1), 64−75. (3) Jinno, J.-i.; Kamada, N.; Miyake, M.; Yamada, K.; Mukai, T.; Odomi, M.; Toguchi, H.; Liversidge, G. G.; Higaki, K.; Kimura, T. Effect of Particle Size Reduction on Dissolution and Oral Absorption of a Poorly Water-Soluble Drug, Cilostazol, in Beagle Dogs. J. Controlled Release 2006, 111 (1−2), 56−64. (4) Müller, R. H.; Peters, K. Nanosuspensions for the Formulation of Poorly Soluble Drugs: I. Preparation by a Size-Reduction Technique. Int. J. Pharm. 1998, 160 (2), 229−237. (5) Rabinow, B. E. Nanosuspensions in Drug Delivery. Nat. Rev. Drug Discovery 2004, 3 (9), 785−796. (6) Choi, J.-Y.; Yoo, J. Y.; Kwak, H.-S.; Uk Nam, B.; Lee, J. Role of Polymeric Stabilizers for Drug Nanocrystal Dispersions. Curr. Appl. Phys. 2005, 5 (5), 472−474. (7) Nutan, M. H.; Reddy, I. General Principles of Suspensions. In Pharmaceutical Suspensions; Kulshreshtha, A. K., Singh, O. N., Wall, G. M., Eds.; Springer: New York, 2010; pp 39−65. (8) Pongpeerapat, A.; Higashi, K.; Tozuka, Y.; Moribe, K.; Yamamoto, K. Molecular Interaction among Probucol/PVP/SDS Multicomponent System Investigated by Solid-State NMR. Pharm. Res. 2006, 23 (11), 2566−2574. (9) Liu, P.; Rong, X.; Laru, J.; van Veen, B.; Kiesvaara, J.; Hirvonen, J.; Laaksonen, T.; Peltonen, L. Nanosuspensions of Poorly Soluble Drugs: Preparation and Development by Wet Milling. Int. J. Pharm. 2011, 411 (1−2), 215−222. (10) Hu, J.; Johnston, K. P.; Williams, R. O. Nanoparticle Engineering Processes for Enhancing the Dissolution Rates of Poorly Water Soluble Drugs. Drug Dev. Ind. Pharm. 2004, 30 (3), 233−245. 1571
DOI: 10.1021/mp500872g Mol. Pharmaceutics 2015, 12, 1564−1572
Article
Molecular Pharmaceutics (31) Sheth, A. R.; Lubach, J. W.; Munson, E. J.; Muller, F. X.; Grant, D. J. W. Mechanochromism of Piroxicam Accompanied by Intermolecular Proton Transfer Probed by Spectroscopic Methods and Solid-Phase Changes. J. Am. Chem. Soc. 2005, 127 (18), 6641− 6651. (32) Sakai, T.; Alexandridis, P. Mechanism of Gold Metal Ion Reduction, Nanoparticle Growth and Size Control in Aqueous Amphiphilic Block Copolymer Solutions at Ambient Conditions. J. Phys. Chem. B 2005, 109 (16), 7766−7777. (33) Tyrrell, Z. L.; Shen, Y.; Radosz, M. Fabrication of Micellar Nanoparticles for Drug Delivery through the Self-Assembly of Block Copolymers. Prog. Polym. Sci. 2010, 35 (9), 1128−1143. (34) Basak, R.; Bandyopadhyay, R. Encapsulation of Hydrophobic Drugs in Pluronic F127 Micelles: Effects of Drug Hydrophobicity, Solution Temperature, and pH. Langmuir 2013, 29 (13), 4350−4356. (35) Alexander, S.; Cosgrove, T.; Prescott, S. W.; Castle, T. C. Flurbiprofen Encapsulation Using Pluronic Triblock Copolymers. Langmuir 2011, 27 (13), 8054−8060. (36) Shin, S.-C.; Cho, C.-W. Physicochemical Characterizations of Piroxicam-Poloxamer Solid Dispersion. Pharm. Dev. Technol. 1997, 2 (4), 403−407. (37) Melosh, N. A.; Lipic, P.; Bates, F. S.; Wudl, F.; Stucky, G. D.; Fredrickson, G. H.; Chmelka, B. F. Molecular and Mesoscopic Structures of Transparent Block Copolymer−Silica Monoliths. Macromolecules 1999, 32 (13), 4332−4342. (38) Flodström, K.; Wennerström, H.; Alfredsson, V. Mechanism of Mesoporous Silica Formation. A Time-Resolved NMR and TEM Study of Silica−Block Copolymer Aggregation. Langmuir 2003, 20 (3), 680−688. (39) Lindfors, L.; Skantze, P.; Skantze, U.; Rasmusson, M.; Zackrisson, A.; Olsson, U. Amorphous Drug Nanosuspensions. 1. Inhibition of Ostwald Ripening. Langmuir 2005, 22 (3), 906−910. (40) Heald, C. R.; Stolnik, S.; Kujawinski, K. S.; De Matteis, C.; Garnett, M. C.; Illum, L.; Davis, S. S.; Purkiss, S. C.; Barlow, R. J.; Gellert, P. R. Poly(lactic acid)−Poly(ethylene oxide) (PLA−PEG) Nanoparticles: NMR Studies of the Central Solidlike PLA Core and the Liquid PEG Corona. Langmuir 2002, 18 (9), 3669−3675. (41) Redenti, E.; Zanol, M.; Ventura, P.; Fronza, G.; Comotti, A.; Taddei, P.; Bertoluzza, A. Raman and Solid State 13C-NMR Investigation of the Structure of the 1:1 Amorphous Piroxicam: βCyclodextrin Inclusion Compound. Biospectroscopy 1999, 5 (4), 243− 251. (42) Xinjuan, L.; Weigui, F.; Yinong, W.; Tiehong, C.; Xiaohang, L.; Hai, L.; Pingchuan, S.; Qinghua, J.; Datong, D. Solid-state NMR Characterization of Unsaturated Polyester Thermoset Blends Containing PEO−PPO−PEO Block Copolymers. Polymer 2008, 49 (12), 2886−2897. (43) Kojima, T.; Higashi, K.; Suzuki, T.; Tomono, K.; Moribe, K.; Yamamoto, K. Stabilization of a Supersaturated Solution of Mefenamic Acid from a Solid Dispersion with EUDRAGIT EPO. Pharm. Res. 2012, 29 (10), 2777−2791. (44) Lubach, J. W.; Xu, D.; Segmuller, B. E.; Munson, E. J. Investigation of the Effects of Pharmaceutical Processing upon Solidstate NMR Relaxation Times and Implications to Solid-State Formulation Stability. J. Pharm. Sci. 2007, 96 (4), 777−787. (45) Sheth, A. R.; Bates, S.; Muller, F. X.; Grant, D. J. W. Polymorphism in Piroxicam. Cryst. Growth Des. 2004, 4 (6), 1091− 1098. (46) Calucci, L.; Forte, C.; Buwalda, S. J.; Dijkstra, P. J. Solid-State NMR Study of Stereocomplexes Formed by Enantiomeric Star-Shaped PEG−PLA Copolymers in Water. Macromolecules 2011, 44 (18), 7288−7295. (47) Göppert, T. M.; Müller, R. H. Protein Adsorption Patterns on Poloxamer- and Poloxamine-Stabilized Solid Lipid Nanoparticles (SLN). Eur. J. Pharm. Biopharm. 2005, 60 (3), 361−372. (48) Bertoluzza, A.; Rossi, M.; Taddei, P.; Redenti, E.; Zanol, M.; Ventura, P. FT-Raman and FT-IR Studies of 1:2.5 Piroxicam: βCyclodextrin Inclusion Compound. J. Mol. Struct. 1999, 480−481 (0), 535−539.
(49) Su, Y.-l.; Wang, J.; Liu, H.-z. FTIR Spectroscopic Investigation of Effects of Temperature and Concentration on PEO−PPO−PEO Block Copolymer Properties in Aqueous Solutions. Macromolecules 2002, 35 (16), 6426−6431. (50) Guo, C.; Liu, H.; Wang, J.; Chen, J. Conformational Structure of Triblock Copolymers by FT-Raman and FTIR Spectroscopy. J. Colloid Interface Sci. 1999, 209 (2), 368−373. (51) Wu, L.; Zhang, J.; Watanabe, W. Physical and Chemical Stability of Drug Nanoparticles. Adv. Drug Delivery Rev. 2011, 63 (6), 456−469. (52) Merisko-Liversidge, E.; Liversidge, G. G.; Cooper, E. R. Nanosizing: a Formulation Approach for Poorly-Water-Soluble Compounds. Eur. J. Pharm. Sci. 2003, 18 (2), 113−120. (53) Cheow, W. S.; Kiew, T. Y.; Yang, Y.; Hadinoto, K. Amorphization Strategy Affects the Stability and Supersaturation Profile of Amorphous Drug Nanoparticles. Mol. Pharmaceutics 2014, 11 (5), 1611−1620. (54) Yang, W.; Johnston, K. P.; Williams, R. O., III. Comparison of Bioavailability of Amorphous versus Crystalline Itraconazole Nanoparticles via Pulmonary Administration in Rats. Eur. J. Pharm. Biopharm. 2010, 75 (1), 33−41.
1572
DOI: 10.1021/mp500872g Mol. Pharmaceutics 2015, 12, 1564−1572
